[7] present the first genome-wide transcriptomic analysis of the circulating hemocytes of the malaria vector Anopheles gambiae following natural infection with the rodent malaria parasit
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Julián F Hillyer
Address: Department of Biological Sciences and Institute for Global Health, Vanderbilt University, VU Station B 35-1634, Nashville,
TN 37235-1634, USA Email: julian.hillyer@vanderbilt.edu
M
Mo ossq qu uiitto oe ess aan nd d m miiccrro ob be ess
Throughout their lifetime, mosquitoes are in danger of
acquiring deadly pathogens During their egg, larval and
pupal stages, mosquitoes live in aquatic environments that
are often rife with bacteria Culex pipiens, for instance, thrive
in sewer systems As adults, mosquitoes often lose their legs,
creating openings by which pathogens can enter their body
Female mosquitoes also engage in the dangerous behavior
of biting vertebrates and ingesting their blood This is done
to acquire the nutrients necessary for the production of
large numbers of eggs, but it exposes mosquitoes to
blood-borne pathogens, such as Plasmodium, filarial nematodes
and arboviruses Besides being deadly and debilitating to
humans, these organisms are pathogenic to mosquitoes if
acquired in high enough numbers
So how does a mosquito respond to a microbial pathogen?
When a foreign invader enters the body cavity of a
mosquito it elicits a systemic immune response Similarly to
that of vertebrates, this immune response has both humoral
and cellular components However, the invertebrate
response lacks the properties of somatic hypermutation and
immune memory that are hallmarks of vertebrate adaptive
immunity The mosquito cellular immune response
includes phagocytosis and encapsulation by hemocytes (blood cells) The humoral response includes the phenoloxidase cascade system of melanization (an enzymatic process in which melanin polymers cross-link with proteins, sequestering pathogens and closing wounds), inducible antimicrobial peptides, reactive oxygen and nitrogen intermediates, and pattern recognition molecules
As with vertebrates, the line between cellular and humoral immunity is blurred because many humoral components are produced by hemocytes Because of their involvement in both cellular and humoral pathways, the circulating nature
of these cells and their ability to respond rapidly to an infection, it is now clear that hemocytes are the first line of defense against microbes that enter the hemocoel (body cavity) of the mosquito [1]
Given their fundamental role in immunity, it is surprising that little is known about the biology of mosquito hemocytes This is probably because they are few in number and are difficult to manipulate Much of what we know comes from studies that have morphologically and functionally characterized hemocyte subpopulations and described their role in pathogen killing and sequestration [2,3] Other studies have focused on the discovery and
A
Ab bssttrraacctt
Mosquito hemocytes are blood cells that are fundamental for combating systemic infection A
study published in BMC Genomics shows that hemocyte gene transcription in response to
immune challenge is pathogen-specific and reaffirms the primary role of these cells in
immunity
Published: 5 June2009
Journal of Biology 2009, 88::51 (doi:10.1186/jbiol151)
The electronic version of this article is the complete one and can be
found online at http://jbiol.com/content/8/5/51
© 2009 BioMed Central Ltd
Trang 2characterization of individual genes and proteins, enabling
in-depth investigations of a limited number of targets that
were initially identified because of homology to genes with
known function in other organisms [4] However, a
con-siderable percentage of the mosquito genes that have been
identified either bioinformatically or through expressed
sequence tag (EST) projects are of unknown function
Because single-gene approaches are unlikely to focus on
these unknowns (many of which may be crucial),
whole-genome transcriptomic and proteomic analyses are needed
to narrow the field
Recent studies have begun to exploit mosquito genomic
data to screen thousands of genes simultaneously for
trans-criptional changes after various treatments Initial work on
mosquito hemocytes has included the characterization of
transcriptional changes in hemocytes from the mosquito
Armigeres subalbatus following infection with the filarial
nematode Brugia malayi and in hemocytes from the
mosquito Aedes aegypti following infection with live bacteria
[5,6] Clearly, additional work is needed in other medically
important vectors to identify genes that are regulated in
response to infection
T
Th he e ttrraan nssccrriip ptto om miicc p prro offiille e o off A An nophelle ess ggaam mb biiaae e
h
he em mo occyytte ess
In a recent article published in BMC Genomics, Baton et al
[7] present the first genome-wide transcriptomic analysis of
the circulating hemocytes of the malaria vector Anopheles
gambiae following natural infection with the rodent malaria
parasite Plasmodium berghei and after immune challenge
with heat-killed Escherichia coli and Micrococcus luteus A
total of 4,047 genes were found to be transcribed in
hemocytes, of which 279 were present in at least two-fold
higher abundance in hemocytes than in the rest of the body
whereas 266 were found in lower abundance Of the
enriched transcripts, only 54.5% have predicted functions,
highlighting the gap in our knowledge of mosquito biology
Of the genes with predicted functions, all components of
the immune response were represented, including pattern
recognition molecules, antimicrobial peptides, serine
pro-teases, serine protease inhibitors, signal transduction proteins,
stress response proteins, melanization-related molecules,
redox/oxidoreductive molecules, and cytoskeletal
organiza-tion and rearrangement (phagocytosis) proteins Immune
challenge with Plasmodium or bacteria resulted in the
differ-ential regulation of 959 genes, of which immunity-related
genes were overrepresented whereas
replication/trans-cription/translation-related genes were underrepresented,
further showing that immune function is the primary role
of hemocytes (Figure 1) When compared with previous
studies, the transcriptome of A gambiae hemocytes is
mostly consistent with the transcriptomic profile of other mosquito species but not with that of Drosophila [6,8], illus-trating evolutionary divergence within the order Diptera and underscoring the importance of directly studying insect species of vectorial significance
D Diiffffe erre en nttiiaall iim mm mu une rre essp ponsse e aaggaaiin nsstt p paatth ho ogge en nss
Mosquitoes mount strong phagocytic immune responses against E coli, whereas sequestration of M luteus is primarily
by melanization [3] Plasmodium ookinetes (the motile zygotes of the parasite) in the midgut are killed by either lysis or melanization within 48 hours of infection [4] In contrast, Plasmodium sporozoites (the infective stage) migrat-ing through the hemocoel durmigrat-ing the third week after infection are killed by mechanisms that have not been firmly characterized However, the low levels of phagocytosis and melanization observed during migration suggest that most parasites are killed by some form of lytic mechanism [9] These differences in the immune responses mounted against different pathogens are in agreement with the data pre-sented by Baton et al [7], which reveal distinct trans-criptional signatures against two different bacterial species and between two stages of malaria parasites After Plasmo-dium berghei infection, a total of 431 genes were differ-entially expressed in hemocytes However, only 5.3% of the
51.2 Journal of Biology 2009, Volume 8, Article 51 Hillyer http://jbiol.com/content/8/5/51
F Fiigguurree 11 Functional classification of genes transcribed in hemocytes Among the genes transcriptionally regulated (up or down) following immune challenge, genes that function in immunity and apoptosis are overrepresented (blue) whereas genes that function in replication, transcription and translation are underrepresented (red) Genes in other functional classes (green) are not regulated at a higher or lower frequency than would be expected if there was no association between functional class and transcriptional regulation following challenge
Hemocytes:
Granulocytes Oenocytoids
Proteolysis
Cytoskeletal/
Structural
Transporters Metabolism
Immunity/
Apoptosis
Redox/Stress/ Mitochondrial
Replication/
Transcription/Translation
Trang 3genes differentially expressed during Plasmodium infection
were regulated in a similar manner for both the ookinete
and sporozoite stages, whereas 3.7% of genes were
regu-lated in opposite directions, indicating that more than 90%
of genes were regulated exclusively during one of the two
infection stages assayed Genes involved in melanization
were induced during ookinete invasion but not during
sporozoite migration, consistent with previous reports that
ookinetes often become melanized but that this rarely
happens to sporozoites [4,9] Interestingly, 37.2% of the
immune genes regulated during sporozoite migration were
members of the fibrinogen-related protein family (FREPs;
also known as FBNs) of mosquitoes This family is made up
of 59 genes in A gambiae, an expansion from the 14 genes
found in Drosophila [10] FBNs in Anopheles and other
mosquitoes have been shown to be involved in antibacterial
and anti-Plasmodium immunity, and it is tempting to
specu-late that their expansion was a consequence of continuous
exposure to blood-borne pathogens
After challenge with heat-killed E coli or M luteus, 641
transcripts were differentially regulated in hemocytes, but
only 6.9% of those transcripts were similarly regulated in
the two groups [7] This was due mainly to a weaker
res-ponse in transcriptional regulation following E coli challenge,
as M luteus altered the transcriptional state of almost four
times as many genes as E coli When only genes with
putative immune function were analyzed, 7.7% of genes
were differentially regulated in a similar manner E coli and
M luteus both induced genes involved in melanization,
even though the latter pathogen was visually observed to
elicit this immune process at a considerably higher rate In
addition, transcripts of genes involved in phagocytosis
either decreased in abundance or were not regulated
follow-ing immune challenge with E coli, whereas transcription of
several genes involved in this immune process increased in
abundance after exposure to M luteus, seemingly in conflict
with the observation that phagocytic events are much more
common against E coli than M luteus It is probable that
this is the result of different molecular interactions during
the internalization of the two pathogens, including the
possible requirement of melanization of M luteus before
the onset of phagocytosis [3]
Overall, the data presented by Baton et al [7] are mostly
consistent with previous transcriptomic analyses of the
hemocytes of other mosquito species [5,6], with the
excep-tion of the level of immune inducexcep-tion in A gambiae
hemocytes following challenge with heat-killed E coli
Possible reasons for these discrepancies include mosquito
species-specific differences or that inoculation with dead
bacteria elicits a weaker response than infection with living
bacteria Furthermore, given that the rodent malaria parasite
Plasmodium berghei and the human malaria parasite Plasmodium falciparum elicit different midgut and carcass transcriptional profiles in response to ookinete invasion [11], future studies will need to address whether the hemocyte response now being reported [7] is similar to the response that occurs during infection with human malaria parasites Nevertheless, the data presented by Baton et al [7] provide a comprehensive dataset that will serve as a starting point for the functional characterization of numerous mosquito genes The report of the breadth of genes transcribed by hemocytes, together with data on their cellular biology, supports the hypothesis that they form the primary component of the mosquito immune response [1-3,7]
A
Ap pp plliiccaattiio on n iin n ttrraan nssm miissssiio on n cco on nttrro oll ssttrraatte eggiie ess
Plasmodium parasites, the causative agents of malaria, kill over a million people per year, and another 500 million people suffer from clinical disease Currently, the control of mosquito-borne diseases has consisted of treating infected individuals, killing the mosquito vector and limiting vector-human contact Although these approaches have reduced disease prevalence, their efficacy is diminishing, mainly because of the emergence of drug resistance by Plasmodium parasites and insecticide resistance in the insect vector Thus, because of the reduced efficacy of current control methods, compounded by the failure to discover new drugs, insecticide replacements and effective vaccines, it has become necessary to develop new control strategies
One possible strategy that has gained support in recent years is to genetically manipulate insect pests such that they are unable to transmit disease-causing pathogens, and to mass release them into the environment to displace natural populations of susceptible mosquitoes Before such a strategy can be implemented several hurdles must be overcome, one of which is the identification of candidate mosquito genes that confer resistance to infection The best candidate genes are probably transcribed in hemocytes, because these cells are involved in immune responses throughout the insect and even produce proteins with anti-parasitic activity in the midgut [4] The study by Baton et al [7] provides a comprehensive dataset of gene transcription following Plasmodium infection and sets the stage for in-depth functional studies on the role of candidate genes in fighting infection
A Acck kn no ow wlle ed dgge emen nttss
The author is funded by NSF grant IOS-0817644
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Re effe erre en ncce ess
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51.4 Journal of Biology 2009, Volume 8, Article 51 Hillyer http://jbiol.com/content/8/5/51